http://www.unc.edu/courses/2009spring/envr/740/001 slide 45Begin 02/03/09Much less is known about eukaryotic origins; however studies on yeast reveal a 50 bp segment of yeast DNA referred to by the acronym ARS (autonomously replicating sequence) which has been shown to contain all the elements required to permit initiation of DNA replication in vitro. The function of ARS has been explored by systematic mutation of the sequence, one base at a time, and the results shown on the overhead, which is a bar graph indicating % loss of activity for mutation at each base.[OH, ARS] At one 11 bp A•T sequence in the “core”, site A, mutation abolishes all origin function; there are several additional close copies of this sequence where mutation reduces origin function. The B elements are binding sites for additional proteins that are involved in assembling the replicon. Regulation of replication in eukaryotic systems is complex and not as well characterized as for prokaryotes. There are both positive and negative controls. The cartoon on the next overhead shows presence of several proteins which are required for initiation of replication. These proteinsenter the nucleus and accumulate during G1 phase, a period in the cell cycle which leads up to DNA replication. (Remember protein synthesis occurs in the cytoplasm.)[OH, licensing factor]Because of their required presence for replication, the proteins are called licensing factors. The ARS sequence is bound at sites A and B1 by a complex of proteins called ORC (origin recognition complex). Initiating a sequence shown on the next overhead.[OH, ORC complex]Cdt1 and Cdc6 bind to DNA, allowing the MCM (mini-chromosome maintenance) complex to bind to DNA during G1 phase, thereby 'licensing' DNA for a single round of replication. MCM 1proceeds along DNA ahead of the replication fork, as depicted on the overhead describing the picture of eukaryotic replication, possibly serving as a helicase. The MCM complex, Cdt1 and possibly Cdc6 are displaced from DNA during S phase. A protein called geminin which is synthesized during S phase, binds to displaced Cdt1 during S, G2 and M phases, preventing re-licensing of DNA replication within the same cell cycle. While events surrounding replication are not known in detail, this is clearly a critically important activity, since release of control of replication is one of the defining characteristics of cell transformation.TRANSLATION OF GENETIC CODE TO FUNCTIONAL PROTEINS.In addition to storing genetic information for perpetuation by replication, DNA also is used to provide the information to assemble proteins, which are required for the cell to function. Will go over very quickly, out of necessity to learn vocabulary and define some features that you will encounter in reading. The synthesis of proteins begins with transcription of DNA, in which a region of DNA, coding for a protein or proteins, is copied into RNA. The DNA-coded information that is required to assemble proteins lies in the sequence of DNA bases on one of theDNA strands, with a group of 3 nucleotides, or triplet, coding for one amino acid. The triplet is also called a codon, and a DNA strand is “read” as a sequence of non-overlapping codons. As you can infer from what I have just said, the DNA itself is not used directly in the assembly of proteins, but is first copied into an intermediate ribonucleic acid called messenger RNA (abbreviated mRNA) which serves as the actual platform for assembly of proteins. Only one strand of the DNA double helix is transcribed. [OH; illustrate sense/antisense mRNA]The DNA strand used as a template is called the anticoding or antisense strand, while the strand not copied, having the same sequence in terms of DNA bases as the RNA strand, is the coding orsense strand. Important to get these terms straight! As a footnote, addition of antisense DNA or fragments of antisense DNA to a cell is one strategy to block the translation of mRNA into proteins and so is used to silence genes in vitro as well as being actively explored by pharmaceutical companies for applications in a variety of therapies. Cells also use short sequences of antisense RNA, called siRNA, or small interfering RNA to tie up mRNA as a 2means for post-transcription regulation of gene expression. The next slide reviews the distinctions between RNA and DNA:Next overhead: RNA and DNA differences(1) the ribose of RNA bears a hydroxy group on the 2′ carbon, (2) the base uracil (U) is substituted for thymine, and (3) RNA normally exists as a single-stranded molecule, rather than adouble helix.Since there are 4 nucleotides that can be arranged in groups of three, and the order of arrangement counts, the number of possible codons is 43= 64. The next descriptions are also important to learn correctly. Codons are always described in terms of the coding sequence (sense sequence), written conventionally from the 5′ to 3′ direction and represented by the RNA bases A,U,G,C. The single-letter codes are always used.Since there are 20 amino acids and 64 codons, it is obvious that there must be considerable redundancy in the code, and this is, in fact, the case. In redundant codons, the base in the third position (3′-end) is variable, as illustrated in the overhead:[OH; use Phe, Leu as examples]The extent of redundancy is related to the frequency of utilization of each amino acid, probably astrategy evolved to minimize coding errors due to mispairings, since it turns out that the pairing at the third position of the codon is “looser” than at positions 1 and 2. Of the 64 codons, 61 represent amino acids. The remaining 3 codons: UAA, UAG and UGA represent signals to terminate protein synthesis, and are therefore called stop codons. In order for proteins to be assembled correctly, it is necessary to establish the correct reading frame; i.e., establish the location of the initial codon for a protein. Translation of RNA usually begins with the codon 5′AUG3′ (methionine) - in prokaryotes, sometimes also GUG (valine).3The process of translation of mRNA into proteins is complex, and we will cover it in a most sketchy manner- mostly to introduce important vocabulary. However, assembly of the proteins isclearly an important aspect of cell function and anyone interested in a more complete description, can read the sections. in Genes IX indicated in the syllabus. The connection between mRNA and the amino acids corresponding to the codons is